Rearing of aquatic species with dha-rich prey organisms

ABSTRACT

A method of rearing aquatic species is provided, comprising feeding the aquatic species during at least part of the larval and/or post-larval stage with prey organisms, such as Aremia or rotifers, having in their total lipid content a DHA content of at least 12 wt %. The method is suitable for the rearing of fish species such as halibut, turbot, bass, bream and flounder.

FIELD OF THE INVENTION

[0001] The present invention is within the field of aquaculture, inparticular there is provided a method for aquacultural rearing ofaquatic species on prey organisms enriched in highly unsaturated fattyacids (HUFAs), in particular docosahexaenoic acid (DHA), providing highsurvival rates during the fish larval stage.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] The consumption of seafood species for which there is a highconsumer demand such as salmon, trout, halibut and eel is increasing anddue to this high demand and limited natural stocks, much effort is spenton developing cost effective aquacultural methods of farming suchspecies. A particularly serious problem is to secure a high survivalrate of the hatched larvae of the species being cultivated.

[0003] Expansion of the aquaculture industry requires that severalproblems be addressed, one of the most significant being the difficultyof supplying live prey organisms which provide a nutritionally adequatefeed for the larvae. Larval fish in the wild consume a mixed populationof phytoplankton prey organisms that provide a balanced nutrition.However, collecting phytoplankton in sufficient quantities to meet thedemand in aquaculture is not feasible. As an alternative, selectedspecies of prey organisms, in particular rotifers and Artemia species,are presently cultivated and used as feed.

[0004] Generally however, such artificially cultivated prey organisms,although they provide adequate amounts of protein and energy, have alipid composition which is not adequate to cover the requirement forcertain HUFAs, in particular DHA and EPA (eicosapentaenoic acid) whichare essential for the optimum survival, growth and development oflarvae. Specifically, it has been shown that a high content of DHA isrequired and that the ratio between DHA and EPA in the prey organismsshould be at least 1:1 and preferably at least 2:1.

[0005] Currently, this problem is being addressed by cultivating theprey organisms in the presence of enrichment compositions permitting theorganisms to be enriched in respect of these essential fatty acids.However, presently available commercial compositions for that purposesuch as products sold under the tradename Selco (TM) do not meet theabove requirements in that the DHA content is relatively low and/or theDHA:EPA ratio is not high enough. Using such compositions Artemiaenrichment levels of 3-5% DHA of total lipids have been reported (McEvoyet al. Aquaculture 163 (1998) 237-250), and 12 to 15% survival rates offish fed such Artemia (McEvoy et al. supra; Navarro et al. J. Fish Biol.43 (1993) 503-515). ). In this context, survival rates are defined assurvival percentage from first feeding through metamorphosis. Forcost-effective aquaculture production a larval survival rate of 50% andpreferably higher should be obtained.

[0006] WO 99/37166 discloses a method for the enrichment of live preyorganisms with nutrients essential for fish larvae based on the use ofdry soap powders of HUFAs obtained from the waste stream of marine algaeoil extraction. Artemia DHA enrichment levels of about 2.7% of dryweight are disclosed, but the use in aquaculture and efficacy withrespect to fish larvae survival is not disclosed.

[0007] Another material intended for use in aquaculture is described inWO 99106585. Examples disclose a DHA content of 24 wt %, but thephospholipid content is not disclosed. The material however, contains ahigh proportion of free fatty acids (about 32-37 wt %) and a highcontent of non-lipid material which may reduce the lipid uptakeefficiency of prey animals. A high content of free fatty acids isgenerally considered harmful for fish juveniles.

[0008] Neither of the two last-mentioned materials is fish-based andthey lack many HUFAs found in fish, such as EPA and other n-3 fattyacids, desirable for fish juveniles.

[0009] In a recent review by Sargent et al. (Aquaculture 179 (1999)217-229) it is emphasized that in addition to the requirement in respectof HUFAs, fish larvae have a dietary requirement for phospholipids andit is stressed that the ideal diet for fish larvae is a diet having acomposition similar to the yolk of the eggs. According to these authorsfish egg yolk contains about 10 wt % (on a dry matter basis)phospholipids which contain about 17 wt % of DHA and about 9 wt % ofEPA. These authors conclude in their review that a problem remains withrespect to how to construct such a diet on a commercial scale fromcurrently available materials.

[0010] It has now been found that it is possible to provide enrichedaquacultural prey organisms having, in respect of HUFAs andphospholipids, a composition which is very close to that of fish eggyolk. By using the prey organisms of the invention it is possible tosecure optimum survival, growth, pigmentation and morphogenesis ofaquatic organism larvae such as halibut larvae. As demonstrated herein,the invention provides much higher survival rates during the larvalstage and increased quality parameters than previously disclosed forfish such as Halibut, thus making aquacultural rearing of manyhigh-demand fish species more economical and commercially viable.

SUMMARY OF INVENTION

[0011] The invention provides a method of rearing an aquatic species,the method comprising feeding the aquatic species during at least partof the larval and/or post-larval stage with prey organisms having intheir total lipid content a content of DHA of at least 12 wt %.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The method according to the invention, of rearing an aquaticspecies, comprises feeding the aquatic species during at least part ofthe larval and/or post-larval stage with prey organisms having in theirtotal lipid content a content of DHA of at least 12 wt %, preferably atleast 15 wt %, such at least 17 wt % more preferably at least 20 wt %,such as at least 25 wt %.

[0013] In preferred embodiments, the prey organisms also contain asignificant amount of phospholipids, such as e.g. in the range of 5-35wt % of the total lipid content, such as in the range of 5-25 wt %including in the range of 10-20 wt % of total lipid.

[0014] It is additionally preferred that the prey organisms provideother HUFAs which are desired for fish larvae and juveniles, such as EPAand other fish-characteristic n-3 HUFAs such as 18:3, 18:4, 20:4, and22:5.

[0015] The inventors have found that such prey organisms for the methodof the invention can be obtained by enrichment with DHA enriched lipidmaterials, preferably materials derived from fish-based sources.

[0016] The prey organisms should preferably not contain too high contentof free fatty acids, as these are generally considered harmful in largeamounts to fish larvae and juveniles. Preferably, free fatty acids areless than about 10 wt % of total lipid of the prey organisms accordingto the method of the invention.

[0017] In the present context, the expression ‘prey organisms’ refers toany marine organism which can be used as live feed for larvae of aquaticspecies which are produced in aquacultural facilities. A general reviewof such prey organisms can be found in Lavens & Sorgeloos (eds.) “Manualon the production and use of live food for aquaculture” published by FAO(1995) which is hereby incorporated by reference. Accordingly, the mostcommonly used prey organisms include several classes and genera ofmicroalgae, rotifers, Artemia, zooplankton including copepods,cladocerans, nematodes, and trochophora larvae.

[0018] As used herein, the term ‘aquatic species’ is to be understood inits broadest sense and comprises both limnobiotic and marine species,including fish species such as salmon, trout, carp, bass, bream, turbot,sea bass, sole, milkfish, gray mullet, grouper, sea bream, halibut,flounder, Japanese flounder, and monkfish; crustaceans such as shrimp,lobster, crayfish and crabs; and molluscs such as bivalves.

[0019] A common feature of these aquatic species is that the life cycleincludes one or more larval stages which may have very specificnutritional requirements and accordingly the provision of live preyorganisms meeting this requirement is an essential factor for successfulaquacultural production. As mentioned above, one such specificrequirement is a high content of the essential fatty acid DHA, the term‘essential’ implying that the prey organisms are not capable of de novosynthesis of such compounds.

[0020] A particular embodiment of the invention involves the rearing ofhalibut species, however, the invention also encompasses other aquaticspecies as, for example, those mentioned above.

[0021] A primary benefit of the invention is that much higher survivalrates for fish larvae can be achieved than have hitherto been reached byany other methods, for species like Halibut and many others, to the bestof our knowledge. As demonstrated in the accompanying examples,consistent survival rates for Halibut during the larval stage of 65-80%and even higher, can be achieved in large scale fish farming accordingto the invention, and other quality parameters such as correctpigmentation are excellent.

[0022] In a useful embodiment of the invention, the prey organisms arefed to the aquatic species such as halibut, at a larval stage,preferably such that at least 30% of the larvae are alive at the end ofthe larval stage, more preferably such that at least 50% are alive atthe end of the larval stage including at least 60%, even more preferablysuch that at least 70% are alive at the end of the larval stage,including that at least 80% are alive at the end of the larval stage.

[0023] Correct pigmentation of the grown cultivated species is animportant feature with respect to market value. A normally pigmentedhalibut has a colored ocular side and a white, non-pigmented blind side.In a preferred embodiment of the invention, at least 70% of halibutjuveniles (newly metamorphosed larvae) show correct pigmentation, morepreferably at least 80%, even more preferably at least 90%, such as 95%,including that essentially all juveniles show correct pigmentation.

[0024] In certain embodiments, the invention provides a method ofrearing aquatic species raised not primarily for consumption, such asornamental fish species and aquarium fish species.

[0025] According to the method, the prey organisms for feeding theaquatic species may be selected from any prey organisms that can beraised and used in aquaculture, in useful embodiments the prey organismsare a crustacean species such as Artemia, Copepoda, Daphnia, or Moinaspecies; a Rotifera species including Brachionus plicatilis, Brachionusrotundiformis, and Brachionus rubens; or a Brachiopoda species.

[0026] The embodiments involving Artemia species are particularlyuseful. The Artemia species can be cultivated and used according to themethod of the invention at a naupliar, metanaupliar, or adult stage.

[0027] In certain embodiments of the invention, the aquatic organismsare reared in a marine environment. A marine environment is used hereinto describe an aqueous medium comprising sea water or simulating seawater, such as an aqueous medium with added salt, e.g., sodium chloride.

[0028] According to the invention, the prey organisms may be fed to theaquatic organisms in any suitable form, e.g., as a compositioncomprising the prey organisms. In a preferred embodiment, the organismsof said composition have a content of DHA of at least 20 wt % of thetotal lipid content of the organisms, more preferably at least 25 wt %of the total lipid content of the organisms, such as at least 30 wt % ofthe total lipid content of the organisms.

[0029] In an embodiment of the invention, the composition comprisesaquatic fish feed organisms having a content of DHA of at least 12 wt %of the total lipid content of the organisms, said composition comprisingan aqueous phase of at least 50 wt %. In a particular embodiment, theaqueous phase of the composition comprises at least 0,5 wt % of sodiumchloride.

[0030] The composition is in other embodiments partially dry, such thatis has a water content less-than 50 wt %, such as at the most 40 wt %,including at the most 25 wt %, such as at the most 10 wt %, including atthe most 5 wt % of water. Such a composition may be in any suitableform, including a powder form, granules, and in the form of flakes.

[0031] As can be inferred from the above, in order for the compositionto provide a significant DHA ration, the total lipid content of the preyorganisms needs to be significant such as, e.g., at least 20 wt % on adry matter basis, including at least 25% dw, and preferably at least 30%dw.

EXAMPLE 1 Preparation of an Enrichment Composition for Fish Larvae PreyOrganisms

[0032] A composition for prey organisms such as Artemia species wasprepared by combining and mixing the following ingredients: TABLE 1.1phospholipid-rich component from squid mantles 9.7 g TG 4010 (TM),Croda, essentially triglycerides 78.0 g w/≈40 wt % DHA vitamin C(ascorbyl palmitate) 8.5 g co-emulsifer, BASF Chremophore A25 (TM) 1.6 gGlucan Macroguard (TM) (immunostimulant) 0.8 g vitamin A (vitamin Apalmitate, 1 mill i.u./g) 0.190 g vitamin E (DL-alpha tocopherolacetate) 0.155 g vitamin B (thiamine hydrochloride) 1.2 g TBHQ(antioxidant) 0.036 g Ethoxyquin (antioxidant) 0.036 g Total 100 g

[0033] The phospholipid-rich component is prepared accordingly:

[0034] Minced squid (150 kg) was added to 300 L of isopropanol and themixture was agitated rather vigorously for 4-6 h and left to standovernight. Subsequently, the mixture was filtered and 300 L of hexanewere added to the filtrate and mixed. This resulted in two phases whichwere allowed to separate. The upper phase, which largely consisted ofhexane and isopropanol was separated and subjected to distillation inseveral rounds in vacuum using a 50 L rotary evaporator to yield a totalof 2.2 kg of a phospholipid enriched fraction as a brown-yellowish waxhaving a phospholipid content of about 65 wt % and a total DHA contentof about 40 wt %.

[0035] The TG 4010 material used as a DHA-rich component in thecomposition is derived from fish oil-based material which is enrichedfor DHA, it comprises 40 wt % DHA, about 10 wt % EPA and about 10 wt %other n-3 HUFAs. The fatty acids are mostly in the form of triglyceridesand the material has a very low free fatty acid content. Other materialshave been tested as sources of a DHA-rich component, such as TG 5010(also from Croda) which has a DHA content of about 50 wt %, andenzymatically synthesized highly DHA-enriched triglycerides.

EXAMPLE 2 Cultivation of Enriched Artemia for Rearing of Aquatic Species

[0036] Artemia cysts were hatched under optimal conditions (in seawater,27-29° C., pH about 8, oxygen content above 4 mg/L). The newly hatchednaupliar Artemia were rinsed and put in 250 L tanks to give a density of200.000/L. Temperature was kept at 25-28° C., oxygen content at 5-6 mg/Land pH buffered at 7.5 with sodium bicarbonate (2 g/L). The tanks wereaerated by passing atmospheric air through perforated hoses at bottom oftanks. Enrichment composition as described in Example 1 was added to thetanks to a concentration of 0,2 g/L and the same amount added 10 hlater. 24 h after the first addition of enrichment composition theArtemia has the following lipid composition (31 % dw (dry weight)lipids, numbers in left-most column refer to the number of carbons anddouble bond in the fatty acids of the lipid components, DHA is 22:6 andEPA 20:5): PL TG FFA Total 16% 76% 8% 100% 14:0 8.8 1.0 3.1 0.8 16:015.0 8.8 36.0 11.1 16:1 2.6 3.2 3.1 2.5 18:0 6.4 2.7 6.3 4.2 18:1 25.215.6 13.0 17.1 18:2 4.2 3.5 1.8 3.3 18:3 13.2 19.2 6.5 14.7 18:4 2.2 3.11.7 2.4 20:1 1.6 1.0 0.0 0.9 20:4 2.8 2.1 0.0 2.2 20:5 12.5 10.2 4.4 9.522:1 0.0 0.0 0.0 22:4 0.0 1.1 0.0 1.2 22:5 0.0 1.0 0.0 1.1 22:6 4.6 20.014.8 18.9 99.0 92.5 90.7 90.0

[0037] The Artemia thus obtained has a highly enriched totalconcentration of DHA and is very suitable for feeding fish larvae suchas halibut larvae according to the invention.

EXAMPLE 3 Cultivation of Artemia with Alternative Enrichment Composition

[0038] Newly hatched Artemia wer placed in 250 L tanks and sameconditions as described in Example 2. The Artemia were fed a lipidcomposition mixed 2 wt % Chremophore A25 emulsifier. The lipidcomposition contained 50 wt % of the phospholipid component of theExample 1 composition; 25 wt % ‘DHA-80’, essentially triglyceridescomprising 80 wt % DHA, synthesized enzymatically from glycerol and DHAfatty acid using lipase from Candida Antarctica (as described in U.S.Pat. No. 5,604,119); and 25 wt % Lysi-22 (TM) (Lysi hf, Iceland), a fishoil with 22 wt % DHA. The feed composition was added to the tanks to aconcentration of 0,2 g/L and the same amount added 12 h later. 24 hafter the first addition of enrichment composition the Artemia has thefollowing lipid composition (34% dw lipids): PL TG FFA Total 25% 72% 3%100% 14:0 0.9 1.1 0.0 1.3 16:0 13.6 10.6 32.0 11.2 16:1 3.3 3.5 3.3 3.418:0 5.8 2.2 10.6 3.3 18:1 26.2 15.4 15.7 15.1 18:2 3.7 2.7 0.0 2.5 18:313.8 15.0 4.4 13.7 18:4 2.7 2.1 0.0 2.2 20:1 1.0 1.9 5.3 2.0 20:4 2.11.7 0.0 1.9 20:5 13.1 8.7 5.0 9.7 22:6 8.4 28.8 23.6 28.0 94.6 93.7100.0 94.1

[0039] The Artemia obtained has a very highly enriched totalconcentration of DHA (9,5% dw) in accordance with the invention as wellas other fish-characteristic n-3 HUFAs, and is thus particularlysuitable for feeding fish larvae such as halibut larvae.

EXAMPLE 4 Use of Enrichment Composition for Cultivating Rotifers(Brachionus plichatilis)

[0040] Rotifers were reared under similar conditions as described inExample 2, they were fed with Isochrysis plankton and yeast and enrichedfor 6 h at 27 C with an enrichment composition as described in Example2, except that Croda 50 was used instead of Croda 40, Croda 50containing about 50 wt % of DHA. The rotifers had the following lipidcomposition (22% dw lipids): PL TG FFA Total 32% 56% 13% 100% 14:0 6.67.8 3.3 6.9 16:0 25.9 4.9 15.2 13.0 16:1 1.9 2.5 1.3 2.2 18:0 3.6 5.72.7 4.7 18:1 4.5 4.5 5.5 4.7 18:2 4.9 0.3 2.0 2.0 18:3 3.1 3.2 1.9 3.018:4 2.2 6.2 2.2 4.4 20:1 1.2 1.9 1.5 1.6 20:4 5.0 2.3 2.2 3.2 20:5 10.114.7 14.8 13.4 22:6 25.6 38.8 40.4 35.2 94.7 92.7 93.0 94.3

[0041] The rotifers obtained have a very high total concentration ofDHA, and a very high phospholipid content and thus and thus are highlysuitable for aquacultural rearing according to the invention.

EXAMPLE 5 Use of HUFA- and Phospholipid-Enriched Artemia forAquacultural Rearing of Halibut

[0042] Halibut larvae were first fed at 230-250° d. (‘° d’:multiplication factor of temperature (° C.) and days since hatching.)Circular rearing tanks were used, either 3,5 or 7 m³. Larvae weregradually acclimatized to a rearing temperature of 11° C. and a lightintensity of 300-500 lux. The larvae were fed Artemia twice per day, inthe morning and in the late aftemoon. The Artemia was enriched with anenrichment composition 24 h before the morning feed, then stored at13-15° C. for another 7-8 h for the afternoon feed. Feed rations wereadjusted to allow for good digestion of the Artemia. Microalgae(Isocrysis sp.) wee added to the rearing water to reduce stress andfacilitate maximum ingestion rates. Slight aeration was applied in thecenter of the tanks to homogenize the water quality and the feedparticles. Slight circular current was acquired with the inflow todistribute the larvae. Water exchange was increased from 1,2 times per24 h in the beginning up to 3,3 times per 24 h in the end. Larvalrearing tanks were cleaned daily.

[0043] Survival rates of over 80% in one tank from start of feed to endof larval stage were observed (90% excluding “gapers”: larvae with jawdeformity), and frequently survival rates between 65 and 75% have beenobserved. On average about 80% of juveniles showed correct pigmentation,but upto 96% correct pigmentation in one tank were observed. Correctpigmentation is defined as a normal pigmentation color on the ocularside and no pigmentation on the blind side. About 65% of juveniles onaverage but up to 80% in one tank showed correct eye migration, that ishaving both eyes on the ocular side. Ongoing experiments indicate thateven higher average survival and pigmentation rates are obtainable.

[0044] The results show that DHA-enriched prey organisms according tothe invention are particularly suitable for the rearing of aquaticspecies such as halibut in terms of high survival rates and quality.

1. A method of rearing an aquatic species, the method comprising feedingthe aquatic species during at least part of the larval and/orpost-larval stage with prey organisms having in their total lipidcontent a content of DHA of at least 12 wt %.
 2. A method according toclaim 1 wherein the DHA content in the total content of lipid is atleast 15 wt %.
 3. A method according to claim 2 wherein the DHA contentin the total content of lipid is at least 20 wt %, including at least 25wt %.
 4. A method according to claim 1 wherein the organisms to becultivated are selected from the group consisting of a crustaceanspecies including Artemia, Copepoda, Daphnia, and Moina species;Rotifera species; and Brachiopoda species.
 5. A method according toclaim 4 wherein the crustacean species is an Artemia species.
 6. Amethod according to claim 5 wherein the Artemia species is cultivated inthe aqueous medium at a naupliar, metanaupliar, or adult stage.
 7. Amethod according to claim 4 wherein the organism is of a Rotiferaspecies or a Brachiopoda species.
 8. A method according to claim 7wherein the Rotifera species is selected from a group includingBrachionus plicatilis, Brachionus rotundiformis, and Brachionus rubens.10. A method according to claim 1 wherein the aquatic species beingreared is of a halibut species.
 11. A method according to claim 10wherein the prey organisms are fed to the halibut species at its larvalstage.
 12. A method according to claim 11 wherein at least 30% of thelarvae being fed are alive at the end of the larval stage.
 13. A methodaccording to claim 12 wherein at least 50% of the larvae being fed arealive at the end of the larval stage.
 14. A method according to claim 13wherein at least 70% of the larvae being fed are alive at the end of thelarval stage.
 15. A method according to claim 1 wherein the aquaticspecies being fed is selected from a group consisting of fish such assalmon, trout, carp, bass, bream, turbot, sole, milkfish, grey mullet,grouper, flounder, sea bass, cod, haddock, Japanese flounder, eel;crustaceans such as shrimp, lobster, crayfish and crabs; molluscs suchas bivalves.
 16. A method according to claim 1 wherein the aquaticspecies being fed is selected from an ornamental fish species and anaquarium fish species.
 17. A method according to claim 1 wherein theaquatic organism is reared in a marine environment.
 18. A methodaccording to claim 1 wherein the aquatic organisms is fed an aquaticfeed composition comprising aquatic prey organisms having a content ofDHA of at least 20 wt % of the total lipid content of the organisms. 19.A method according to claim 18 wherein the fish is fed a compositioncomprising aquatic prey organisms having a content of DHA of at least 25wt % of the total lipid content of the organisms.
 20. A method accordingto claim 1 wherein the total lipid content of the feed organisms is atleast 20 wt % on a dry matter basis.
 21. A method according to claim 1wherein the fish is fed a composition comprising aquatic fish feedorganisms having a content of DHA of at least 12 wt % of the total lipidcontent of the organisms, the composition comprising an aqueous phase ofat least 50 wt %.
 21. A method according to claim 1 wherein the fish isfed a composition comprising aquatic fish feed organisms having acontent of DHA of at least 12 wt % of the total lipid content of theorganisms, the composition comprising an aqueous phase of at least 50 wt%.
 22. A method according to claim 21 wherein the aqueous phasecomprises at least 0.5 wt % of NaCl.
 23. A method according to claim 1wherein the fish is fed a composition comprising aquatic feed animalshaving a content of DHA of at least 12 wt % of the total lipid contentof the organisms, the composition having a water content that is lessthan 50 wt % including at the most 10 w %.
 24. A method according toclaim 23 wherein the composition is in a form selected from the groupconsisting of powder, granules and flakes.